从薄荷（Mentha haplocalyx Briq.）叶cDNA中克隆到1个参与精油合成的柠檬烯-6-羟化酶基因，命名为MhL6OH，编码的蛋白质为MhL6OH。序列分析结果表明：该基因蛋白质编码区（CDS）全长1 479 bp，编码492个氨基酸残基； MhL6OH蛋白的理论相对分子质量为55 855.69，理论等电点为pI 8.71，其二级结构中α-螺旋、无规则卷曲、延伸链和β-转角的比例分别为51.02％、30.49％、12.40％和6.10％，且该蛋白质含有保守的细胞色素P450结构域，说明薄荷MhL6OH蛋白属于CYP71家族的D亚家族。多重序列比对结果表明：薄荷MhL6OH蛋白与椒样薄荷（M. piperita Linn.）MpL6OH蛋白和留兰香（M. spicata Linn.）MsL6OH蛋白的氨基酸序列高度相似，均具有细胞色素P450的血红素结合区；并且，MhL6OH蛋白与MpL6OH蛋白底物识别位点（SRS）的序列相似性更高，二者的SRS2、SRS3、SRS5和SRS6序列完全相同，仅分别在SRS1和SRS4序列上存在1个氨基酸差异，说明MhL6OH蛋白与MpL6OH蛋白可能具有相似的底物识别特异性。系统进化树分析结果表明：薄荷与椒样薄荷的亲缘关系最近。组织表达特性分析结果显示：MhL6OH基因的相对表达量在薄荷叶中最高，并远高于其在茎和根中的相对表达量。原核表达分析结果显示：MhL6OH蛋白能够在大肠杆菌〔Escherichia coli （Migula） Castellani et Chalmers〕中高量表达，且其表达量随诱导时间延长而逐渐增大。研究结果显示：薄荷MhL6OH基因组织表达模式与其精油分布一致，MhL6OH蛋白底物识别位点的特异性可能是薄荷醇为薄荷精油主要成分的重要原因。

A limonene-6-hydroxylase gene involved in essential oil biosynthesis was cloned from cDNA of leaf of Mentha haplocalyx Briq.， which was named as MhL6OH， and its encoded protein was MhL6OH. The sequence analysis result shows that the whole length of coding sequence region （CDS） of this gene is 1 479 bp， which encodes 492 amino acid residues. Theoretical relative molecular mass of MhL6OH protein is 55 855.69， and its theoretical isoelectric point is pI 8.71. In its secondary structure， percentages of α-helix， random coil， extended strand， and β-turn are 51.02％， 30.49％， 12.40％， and 6.10％， respectively， and this protein contains conserved cytochrome P450 domain， indicating that MhL6OH protein of M. haplocalyx belongs to D subfamily of CYP71 family. The multiple sequence alignment result shows that amino acid sequences of MhL6OH protein of M. haplocalyx， MpL6OH protein of M. piperita Linn.， and MsL6OH protein of M. spicata Linn. are highly similar， and all of them contain heme binding region of cytochrome P450； moreover， the sequence similarity of substrate recognition site （SRS） between MhL6OH protein and MpL6OH protein is higher， and their SRS2， SRS3， SRS5， and SRS6 sequences are completely identical， while there is only one amino acid difference in SRS1 and SRS4 sequences， respectively， indicating that MhL6OH protein and MpL6OH protein may have similar substrate recognition specificity. The phylogenetic tree analysis result shows that the genetic relationship between M. haplocalyx and M. piperita is the closest. Tissue expression characteristics analysis result shows that the relative expression level of MhL6OH gene in leaf of M. haplocalyx is the highest and much higher than that in stem and root. The prokaryotic expression analysis result shows that MhL6OH protein can highly express in Escherichia coli （Migula） Castellani et Chalmers， and its expression level gradually increases with the elongation of induction time. It is suggested that the tissue expression pattern of MhL6OH gene of M. haplocalyx is consistent with its essential oil distribution， and the specificity of substrate recognition site of MhL6OH protein may be the important reason for menthol as main component of essential oil of M. haplocalyx.